In recent years, the possibilities of therapeutic intervention in sepsis have been augmented by the development biological therapeutic agents that modulate the systemic activation of the inflammatory and coagulatory systems. Two such therapeutic agents are human APC, prepared by recombinant DNA technology, and antithrombin, purified from human plasma. Both agents show anti-coagulatory and anti-inflammatory activity in in vitro test systems or animal models. However, the results of treating unselected patients with sepsis with these agents have been either marginal (APC) or undetectable (antithrombin) in terms of increasing survival. In the case of APC, a beneficial effect on survival was seen in only the group of patients with severe sepsis and an APACHE II score of 25 or more (Bernard et al., 2001). APACHE II is a severity-of-disease classification system which uses a point score based upon initial values of 12 routine physiologic measurements, age, and previous health status to provide a general measure of severity of disease, resulting in a score ranging from 0 to 71 that correlates with the risk of hospital death (Knaus et al., 1985).
The problem is therefore how to improve the selection of patients with sepsis for treatment with APC so that those patients who are most likely to benefit from this treatment receive it, and those who are unlikely to benefit do not. The latter consideration is important, because the administration of APC is both costly and associated with unwanted effects, in particular with an increased incidence of serious bleeding events, including intracranial hemorrhage. While selecting patients for treatment by means of the APACHE II score goes some way towards such a selection, it is the purpose of the present invention to provide an improved method of selecting patients for APC treatment based on demonstrating the patient's requirement for APC by measuring an insufficient activation of endogenous PC. This may select patients for treatment although they have an APACHE II score <25 and may also exclude patients with an APACHE score ≧25 because they already show an appropriate level of activation of endogenous PC.
PC is a protein synthesized in a vitamin-K-dependent manner by the liver as a single polypeptide chain of 461 amino acid residues. It is cleaved into a light chain of 155 amino acid residues linked by a single disulfide bridge to a heavy chain of 262 amino acid residues, both chains being glycosylated. The protein is a serine protease zymogen with an active site in the heavy chain protected by an N-terminal peptide, which is split off during activation. Mature PC has a molecular mass of approximately 62 kDa and is approximately 23% glycosylated. Its concentration in plasma is 3-5 μg/mL and its half-life in the circulation is 6-8 hours. PC is slowly activated by thrombin and 1000-fold more rapidly activated by thrombin in complex with the receptor, thrombomodulin, at the vascular surface of endothelial cells. Here the activation of PC is further 20-fold enhanced by the binding of PC to the endothelial PC receptor (EPCR). PC can also be activated pharmacologically by serine proteases from Agkistrodon snake venom.
APC generated by activation of PC is a serine protease which splits blood coagulation factors Va and VIIIa, using protein S and blood coagulation factor V as cofactors. It thereby limits blood coagulation in the vicinity of membrane-bound factors Va and VIIIa. APC may also have an indirect fibrinolytic activity by inhibiting plasminogen activator inhibitor-1 and by limiting the generation of activated thrombin-activatable fibrinolysis inhibitor. APC may also have an anti-inflammatory action by inhibiting the production of tumor necrosis factor by monocytes, by blocking the adhesion of leukocytes to selecting, and by limiting thrombin-induced endothelial inflammatory responses. In addition, it has been demonstrated to have an inhibitory effect on apoptosis.
APC has a molecular mass of approximately 55 kDa and its normal concentration in plasma has been estimated as lying within the range of 1-3 ng/mL (1.19 ±standard deviation 0.41 ng/mL). The half-life of APC in the human circulation is short, being variously estimated as 20 minutes (Dahlback and Villoutreix, 2003) or 45 minutes (Macias W L et al., 2002). During constant intravenous infusion a steady state concentration is reached after 2 hours, and when infusion is stopped, the concentration of APC in plasma falls to below 10 ng/mL within 2 hours. Endogenous plasma concentrations of APC measurable by the hitherto used immunoenzymatic assay (Gruber and Griffin, 1992) exceed the detection limit normally achieved in practice (10 ng/mL) in only 3.3% of patients with sepsis.
As soon as APC is produced within the circulation, it undergoes inactivation by reacting with serpins (serine protease inhibitors) also present in the blood. These include PCI, alpha1-antitrypsin (AAT), alpha2-antiplasmin and C1-esterase inhibitor (in descending order of second order rate constants). Of these, PCI appears to be of greatest physiologic importance. PCI is a single-chain protein of molecular mass about 57 kDa, and its normal concentration in plasma is about 5 μg/mL. Its half-life in the circulation, measured in rabbits, is about 24 hours (Laurell et al., 1990). APC-PCI complexes are formed with a second order rate constant of 13×103 M−1s−1 (Marlar R A et al., 1993) and their half-life in the circulation is about 20 minutes in rabbits (Laurell et al., 1990), 40 minutes in baboons (Espana F et al., 1991). AAT is a single chain protein of molecular mass about 54 (50-60) kDa, and its normal concentration in plasma is about 2.5 mg/mL, about 500-fold higher than that of PCI. Its half-life in the circulation measured in rabbits is 62 hours (Laurell et al., 1990). APC-AAT complexes are formed with a second order rate constant of 15 M−1s−1 (Marlar et al., 1993) and their half-life in the circulation is 72 minutes in rabbits (Laurell et al., 1990), 140 min in baboons (Espana et al., 1991).
It will be seen that the rate constant for APC-AAT complex formation is only about one thousandth of that for APC-PCI complex formation, but this is partially compensated for by the 500-fold higher concentration of AAT than PCI in plasma. At the same time, the half-life of APC-AAT complexes in the circulation is 3- to 4-fold longer than that of APC-PCI complexes. This results in higher concentrations of APC-AAT complexes in plasma than APC-PCI complexes.
APC can also form complexes with alfa2-antiplasmin, with a second order rate constant of 410 M−1s−1, or very slowly with C1-esterase inhibitor (second order rate constant of <6 M−1s−1). The possible formation of these complexes is not regarded as being physiologically significant.
The median plasma concentration of APC-PCI complex, from blood collected from healthy volunteers into 5-ml vacuum tubes containing 0.5 mL of 0.5 M citrate buffer, pH 4.3, to prevent further complex formation after sampling, is 0.13 ng/mL, with a range of 0.07-0.26 ng/mL (Strandberg et al., 2003). In 18 patients with sepsis the mean concentration of APC-PCI complex in plasma (containing 50 mM benzamidine as an inhibitor of further complex formation) was 3 ng/mL±standard deviation (SD) 2 ng/mL, estimated by enzyme-linked immunosorbent assay (Alcaraz et al., 1995). The mean concentration of APC-AAT complex in these patients was 26 ng/mL±SD 15 ng/ml. The mean level of PC in plasma on day 1 was 69%±SD 28% of the mean normal control value, and the mean level of PCI was 33%±22% of the control value, showing consumption of these components due to activation of PC in sepsis.
Patients with severe sepsis vary markedly in their ability to generate APC (Liaw et al., 2004). The endothelial receptors essential for efficient activation of PC, thrombomodulin and EPCR may be down-regulated in sepsis. For example, the levels of thrombomodulin and EPCR are reduced in the endothelium of children with severe meningococcal sepsis (Faust et al., 2001), and in vitro studies have shown that thrombomodulin and the EPCR are down-regulated by inflammatory cytokines (Esmon, 2004). The extent of this down-regulation is variable, and this means that some patients with sepsis show low levels of endogenous APC as measured by an improved version of the immunoenzymatic assay, a phenomenon that does not correlate with the APACHE II score (Liaw, 2004).